1. Field of the Invention
The present invention relates generally to scanning confocal microscopes and, more particularly, to laser-scanning confocal microscopes that scans a sample with ultraviolet light and detect the resulting fluorescence.
2. Description of Related Art
In the scientific fields of physiology, cytobiology, etc., it is important to investigate the behavior of the intracellular ions of calcium, sodium, magnesium, etc. This is because these ions are thought to be closely linked with intracellular physioactivation. As part of a method of research on the behavior of the intracellular ions, fluorophores commonly are injected into cells. Such fluorophores combine uniquely with the certain species of ions within the cells, and fluoresce when irradiated with excitation light of specified wavelength.
By way of example, fluorescent probes indo-1, fura-2, fluo-3 and rhod-2 are known fluorophores useful in the detection of the calcium ions. Any of these fluorescent probes can be used to detect the presence of calcium ions within the cells. For example, the probe indo-1 fluoresces at wavelengths of either 405 nanometers (nm) or 485 nm in accordance with the concentration of the calcium ions, in response to excitation by ultraviolet radiation having a wavelength of about 350 nm. The probe fura-2, on the other hand, fluoresces at a wavelength of about 500 nm, in response to excitation by ultraviolet radiation having a wavelength of about 340 nm or 380 nm.
When the fluorescent probe has combined with the calcium ions and is excited by the ultraviolet light, it fluoresces in an amount that varies in accordance with the calcium ion concentration. Therefore, the concentration 5 of the calcium ions in each local area of a sample can be determined by measuring the intensity of the fluorescent light. The excitation and fluorescence detection can be carried out across sample surface, whereby a two-dimensional video image can be obtained. Further, a plurality of video images can be obtained in time series, whereby the time behavior of the ions can be investigated in detail.
Where the ratio between the intensities of the two peaks of the fluorescence spectra is detected under the ultraviolet excitation of the fluorescence probe indo-1, or where the ratio between the intensities of the respective peaks of the fluorescence spectra is detected under the alternate excitation operations of the fluorescent probe fura-2 with the two wavelengths of ultraviolet radiation, an accurate measurement of calcium ion concentration can be reliably obtained.
It is desirable to the scan the doped sample with an incident laser beam in a raster pattern and to produce from the resulting fluorescent radiation a standard video-format signal so that fluorescence of the sample can be viewed on a video display. For a true two-dimensional representation of the sample, the successive scans must be linear. It is known to linearize such scans by generating a reference beam that scans at the same rate as the incident laser beam and by directing that reference beam through a Ronchi grating to produce a pulsed beam from which a clock signal can be derived representative of the scanning speed. Although such a technique is effective in roughly linearizing non-linear scan data, the reference beam and incident laser beam do not always scan at rates that precisely track each other.
It should therefore be appreciated there is a need for a scanning confocal microscope that scans a sample in a raster scan pattern in which the scan data is a true representation of the sample's two-dimensional proportions. The present invention fulfills this need.